Detecting specific gene sequences in clinical samples that are associated with disease states or biological conditions is frequently hindered by the low copy number of these gene sequences in the sample. The ability to replicate these gene sequences to improve sensitivity has revolutionized modem molecular genetics. There are currently many different methods for amplifying nucleic acids in samples to improve assay sensitivity, such as: polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202); ligase chain reaction (LCR); nucleic acid sequence-based amplification (NASBA) (U.S. Pat. Nos. 5,409,818 and 5,554,517); strand displacement amplification (SDA); and transcription-mediated amplification (TMA).
Many of these methods are capable of providing more than one billion copies of a single target nucleic acid in a very short time. Accordingly, one of the principle problems of using amplification technologies is that they are susceptible to contamination by exogenous nucleic acids. Although the latter can be controlled using careful laboratory techniques, the former source of contamination is hard to avoid in laboratories that repetitively amplify the same target sequences. In either case, this exogenous nucleic acid may be amplified along with the target nucleic acid in a clinical sample, which may lead to erroneous results.
Many different protocols have been developed in the past several years to prevent carryover contamination. Some of these protocols involve chemical, photochemical and/or enzymatic methods to inactivate the amplicons to prevent them from serving as templates in subsequent amplification reactions. When such methods are combined with appropriate laboratory techniques, the frequency of contamination-associated false-positive results is reduced. Since many of these types of decontamination protocols interfere with the amplification reaction, they must be carried out after amplification has been completed.
One of the more recently described methods for preventing contamination involves the use of UV irradiation to photochemically modify amplicon nucleotide bases. Such irradiation in the presence of certain isopsoralen derivatives forms cyclobutane adducts with pyrimidine bases, and the nucleic acids with these modified bases are no longer capable of serving as templates for subsequent PCR (G. D. Cimino et al., Nucleic Acid Research, 19(l):99-107 (1990)). However, this method has been described as only being useful when carried out after amplification has been completed, since these base reactions are non-specific and the reactants may interfere with the integrity of the target nucleic acid and other reaction components (R. Y. Walder et al., Nucleic Acids Research, 21(18):4339-4343 (1993)). Moreover, most of these currently used methods are adapted for use in PCR. Thus, it is not well established that such methods are equally as effective in other types of amplification reactions. Nor have decontamination protocols been specifically designed to be carried out during any stage of the amplification reaction.
Detection of the amplified nucleic acids involves the use of a labeling compound or compounds that can be measured and quantified. Many such labeling compounds are well known in the art. However, every additional step in the amplification reaction introduces additional reagent costs and assay time. Recently, methods have been develop that allow for simultaneous labeling and decontamination using reagents that are capable of serving both purposes.
Accordingly, there is a need to provide for improved decontamination reagents and protocols that are adapted for use before the amplification reaction has been completed, and that are suitable for simultaneous decontamination and labeling.